1. Field of the Invention
The invention relates to a process for the measurement of the blood circulation of organ and tissue specimens by carrying out the following process steps:
1. Coloration of small plastic spheres PA1 2. Introduction of the plastic spheres into the blood circulation PA1 3. Sampling of a plurality of tissue and/or blood specimens PA1 4. Determination of the number of plastic spheres in the specimens sampled PA1 5. Determination of blood circulation using the values obtained by step 4. PA1 A) Use of monodisperse microspheres PA1 B) Homogeneous coloration of the microspheres PA1 C) Isolation of the microspheres present in the individual specimens by dissolving the tissue or blood portions of the specimen, which leaves the microspheres and their dye content almost unchanged, followed by separating off the liquid phase PA1 D) Determination of the number of microspheres in the specimen by eluting the dye and subsequent determination of the amount of dye eluted.
Furthermore, the invention relates to a colouring process for the coloration of the microspheres used in the abovementioned process.
2. Description of the Prior Art
The known principle of measuring blood circulation is based on the injection of labelled plastic spheres (hereinafter also called microspheres) into the bloodstream of a laboratory animal, subsequent sampling of blood or tissue specimens and determination of the number of microspheres in the individual specimens. Blood circulation of the specimens tested at the time of injection of the microspheres can then be determined from the concentration of the microspheres in the tissue and blood specimens and the injected amount of microspheres.
Implementation of this process using radioactively labelled microspheres is known. Labelling takes place by incorporating various radioactive isotopes into the material comprising the spheres. Although this variant of labelling has been established as a standard method, it has nevertheless some serious disadvantages. The use of radioactive labels forces the user in particular to observe the various radiation protection laws. Thus, the laboratories in which measurements of blood circulation are carried out in this manner must be equipped accordingly and contamination of the persons involved in the tests must be avoided. Likewise, the laboratory animals must be housed in suitably controlled stables. This requires extensive structural measures and thus restricts the number of laboratories in which this process can be carried out. A further factor which makes the known process more expensive is that the animal carcasses contaminated in the course of this process must be stored in decay units before they can ultimately be disposed of. The isotopes usual for labelling are also expensive and rare, and their half lives, some of which are short, limit the use of the process in some cases. The apparatuses for measuring radioactivity (gamma counters, and the like) are also relatively complicated and expensive. In addition, the radioactively labelled microspheres have a density (1.3 g/ml) which differs from the density of the blood, resulting in unphysiological distribution of the microspheres in the bloodstream.
A process in which the radioactively labelled microspheres are replaced by those coloured with a colorant is described by European Patent Application EP 0,194,517 A1. In the process described, the microspheres are coloured in a solution comprising the salt of fat-soluble dyes and chloroform. Evaluation takes place by visual counting, in which the microspheres are counted either directly in the specimen or after being separated from the specimen and introduced into a special counter, a haemacytometer. This visual analytical process makes the method extremely work-intensive and prone to errors and prevents automation of the analysis. In addition, the process has further disadvantages. Thus the diameter of individual spheres from a specimen having a certain nominal diameter deviates by up to 50% from this nominal diameter. An error of this magnitude has an adverse effect on the precision of the entire measurement.
The object was therefore to improve the process for the measurement of blood circulation with the use of coloured microspheres such that measurement can be carried out rapidly, accurately and without large expenditure, in particular without employing large numbers of personnel. The object was in particular to improve the process for determining the number of microspheres in the tissue specimens.